The endocannabinoid system (ECS) is an endogenous modulatory system involved in a wide variety of physiological processes, including learning, thinking, immune, metabolic and reproductive systems. 2- arachidonoylglycerol (2-AG) is a lipid messenger of the ECS, full agonist at Cannabinoid Receptors type 1 (CB1) and type 2 (CB2). 2-AG biosynthesis is catalyzed by diacylglycerol lipase (DAGL), a serine hydrolase that cleaves the ester bond at the sn1 position of 2-arachidonoyldiacylglycerol, releasing 2-AG and a fatty acid. There are two known DAG lipases in humans, DAG lipases ? and ?. Very little is known about the structure and biochemistry of these key ECS enzymes, the only known four helical pass transmembrane serine hydrolases in mammals. Determining the biochemical properties and three dimensional structures of DAG lipases will help us understand their tertiary and quaternary structures, catalytic mechanisms, and substrate binding pocket chemistry and architecture. We propose to use the biochemical and crystallographic tools at our disposal to investigate the structure and biochemistry of DAG lipase ?. First, we have developed and will continue to optimize, an overexpression, solubilization, and purification protocol for recombinant DAGLB? using a mammalian cell expression system. Second, we propose to investigate the oligomeric state of DAGL?, both in solution and in the membrane, using size exclusion chromatography, light scattering, and crosslinking methods. We will also investigate the biochemical properties of DAGL?, determining kinetic parameters using fluorescence and LC-MS assays available to us, testing a panel of candidate substrates, inhibitors, and activators proposed in the literature. Finally, we propose to solve the first crystal structure of a full lengh DAG lipase, DAGL?, using in meso lipidic cubic phase methods, in close collaboration with Dr. Ray Stevens' group. The knowledge on DAG lipases obtained through the work proposed in this project has the potential to become an important tool for the rational design of inhibitors against these key therapeutic targets.